title: ROCK inhibition enhances aggrecan deposition and suppresses matrix metalloproteinase-3 production in human articular chondrocytes.
abstract: Homeostasis of articular cartilage is maintained by a balance between catabolism and anabolism. Matrix metalloproteinase-3 (MMP-3) catabolism of cartilaginous extracellular matrix (ECM), including aggrecan (AGN), is an important factor in osteoarthritis progression. We previously reported that inhibition of Rho-associated coiled-coil forming kinase (ROCK), an effector of Rho family GTPases, activates the chondrogenic transcription factor SRY-type high-mobility-group box (SOX) 9 and prevents dedifferentiation of monolayer-cultured chondrocytes. We hypothesized that ROCK inhibition prevents chondrocyte dedifferentiation by altering the transcriptional balance between MMP-3 and AGN. Normal human articular chondrocytes were cultured in the presence or absence of ROCK inhibitor (ROCKi, Y-27632). Expression of MMP-3 and AGN during monolayer cultivation was assessed by quantitative real-time PCR and western blot analysis. Chondrogenic redifferentiation potential of ROCKi-treated chondrocytes was evaluated by immunohistological analysis of pellet cultures. ROCKi treatment suppressed MMP-3 expression in monolayer- and pellet-cultured chondrocytes but increased AGN expression. Chromatin immunoprecipitation revealed that the association between transcription factors E26 transformation specific (ETS)-1 and SOX9 and their target genes MMP-3 and AGN, respectively, was affected by ROCKi treatment. ROCKi decreased the association between ETS-1 and its binding sites on the MMP-3 promoter, whereas ROCKi promoted the interaction between SOX9 and the AGN promoter. Our results suggest that ROCK inhibition may have an important role in modulating the balance between degradation and synthesis of cartilaginous ECM, a finding that may facilitate development of techniques to prepare differentiated chondrocytes for cartilage regeneration therapy.
authors.affiliation: Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences , Kitaku, Okayama , Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Furumatsu
authors.forename: Emi
authors.initials: E
authors.lastname: Matsumoto-Ogawa
authors.forename: Takaaki
authors.initials: T
authors.lastname: Tanaka
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Lu
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Ozaki
mesh_terms: D000293:Adolescent; D000328:Adult; D053674:Aggrecans / Q000378:metabolism*; D000577:Amides / Q000494:pharmacology*; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002461:Cell Line, Transformed; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008297:Male; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis*; D011725:Pyridines / Q000494:pharmacology*; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D053674:Aggrecans; D000577:Amides; D004791:Enzyme Inhibitors; D011725:Pyridines; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C108830:Y 27632; D054460:rho-Associated Kinases; C511768:MMP3 protein, human; D019278:Matrix Metalloproteinase 3